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1.
Stem Cell Rev Rep ; 15(4): 590-600, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30879244

RESUMO

Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.

2.
J Vis Exp ; (114)2016 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-27683961

RESUMO

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

3.
J Cell Biol ; 211(2): 359-72, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26504171

RESUMO

Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1-infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150(Glued), and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150(Glued), hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1-infected macrophages, leading to strong alterations in phagolysosome biogenesis.


Assuntos
HIV-1/imunologia , Macrófagos/imunologia , Microtúbulos/metabolismo , Fagocitose/imunologia , Salmonella typhimurium/imunologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Células HeLa , Humanos , Proteínas com Domínio LIM/metabolismo , Macrófagos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo
4.
Mol Cytogenet ; 7(1): 59, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25320640

RESUMO

BACKGROUND: Roberts syndrome (RBS) is a rare autosomal recessive disorder mainly characterized by growth retardation, limb defects and craniofacial anomalies. Characteristic cytogenetic findings are "railroad track" appearance of chromatids and premature centromere separation in metaphase spreads. Mutations in the ESCO2 (establishment of cohesion 1 homolog 2) gene located in 8p21.1 have been found in several families. ESCO2, a member of the cohesion establishing complex, has a role in the effective cohesion between sister chromatids. In order to analyze sister chromatids topography during interphase, we performed 3D-FISH using pericentromeric heterochromatin probes of chromosomes 1, 4, 9 and 16, on preserved nuclei from a fetus with RBS carrying compound heterozygous null mutations in the ESCO2 gene. RESULTS: Along with the first observation of an abnormal separation between sister chromatids in heterochromatic regions, we observed a statistically significant change in the intranuclear localization of pericentromeric heterochromatin of chromosome 1 in cells of the fetus compared to normal cells, demonstrating for the first time a modification in the spatial arrangement of chromosome domains during interphase. CONCLUSION: We hypothesize that the disorganization of nuclear architecture may result in multiple gene deregulations, either through disruption of DNA cis interaction -such as modification of chromatin loop formation and gene insulation - mediated by cohesin complex, or by relocation of chromosome territories. These changes may modify interactions between the chromatin and the proteins associated with the inner nuclear membrane or the pore complexes. This model offers a link between the molecular defect in cohesion and the complex phenotypic anomalies observed in RBS.

5.
Dev Cell ; 23(5): 954-67, 2012 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-23153494

RESUMO

The protein Bcl10 contributes to adaptive and innate immunity through the assembly of a signaling complex that plays a key role in antigen receptor and FcR-induced NF-κB activation. Here we demonstrate that Bcl10 has an NF-κB-independent role in actin and membrane remodeling downstream of FcR in human macrophages. Depletion of Bcl10 impaired Rac1 and PI3K activation and led to an abortive phagocytic cup rich in PI(4,5)P(2), Cdc42, and F-actin, which could be rescued with low doses of F-actin depolymerizing drugs. Unexpectedly, we found Bcl10 in a complex with the clathrin adaptors AP1 and EpsinR. In particular, Bcl10 was required to locally deliver the vesicular OCRL phosphatase that regulates PI(4,5)P(2) and F-actin turnover, both crucial for the completion of phagosome closure. Thus, we identify Bcl10 as an early coordinator of NF-κB-mediated immune response with endosomal trafficking and signaling to F-actin remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , NF-kappa B/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fator de Transcrição AP-1/metabolismo , Actinas/metabolismo , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteína 10 de Linfoma CCL de Células B , Linhagem Celular , Endossomos/metabolismo , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , Fagocitose , Receptores Fc/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
6.
Hepatology ; 56(3): 861-72, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22454196

RESUMO

UNLABELLED: The development of human cultured hepatitis C virus (HCV) replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however, this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high-titer infectious virus as well as Japanese fulminant hepatitis type 1, H77/C3, and Con1/C3. This experimental model was employed to demonstrate HCV neutralization or HCV inhibition, in a dose-dependent manner, either by cluster of differentiation 81 or envelope protein 2-specific antibodies or convalescent serum from a recovered HCV patient or by antiviral drugs. CONCLUSION: This new ex vivo model represents a powerful tool for studying the viral life cycle and dynamics of virus spread in native tissue and also allows one to evaluate the efficacy of new antiviral drugs.


Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Fígado/virologia , Replicação Viral , Adulto , Humanos , Técnicas de Cultura de Tecidos , Replicação Viral/efeitos dos fármacos
7.
J Immunol Methods ; 378(1-2): 51-5, 2012 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-22349126

RESUMO

Studying the immunological processes taking place during the initial steps of acute hepatitis C virus (HCV) infection has been a challenge in patients. Shin et al. have recently reported that delayed induction, not impaired recruitment of specific CD8(+) T cells, causes the late onset of acute hepatitis C in chimpanzees (Gastroenterology, 2011). However, further elucidation of the underlying mechanisms is difficult in vivo. We made observations consistent with their conclusions in human liver slices inoculated ex vivo with HCV produced in cell culture (HCVcc). Autologous immune cells were purified from blood and differentially stained prior to their incubation with the slices for 2 hours. A two-photon confocal microscopic analysis revealed that many more stained dendritic and T cells contracted interactions within two-day infected slices than non-inoculated ones (p<0.001). While in the first instance some dendritic and T cells entered into closer interactions, they never did in the latter case. These results suggest that ex vivo infection of human liver slices with HCVcc may be useful for gaining experimental insight regarding the immunological processes taking place at early steps of HCV infections.


Assuntos
Células Dendríticas/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Fígado/imunologia , Linfócitos T/imunologia , Técnicas de Cultura de Células , Células Dendríticas/virologia , Humanos , Fígado/virologia , Ativação Linfocitária/imunologia , Linfócitos T/virologia
8.
Biol Cell ; 104(1): 34-47, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22188458

RESUMO

BACKGROUND INFORMATION: The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. RESULTS: Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. CONCLUSIONS: The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites.


Assuntos
Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Família Multigênica , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
9.
J Vis Exp ; (53): e3054, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21775968

RESUMO

Naïve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay, originally set up by neurobiologists and transposed recently to murine thymus. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.


Assuntos
Linfonodos/citologia , Microscopia Confocal/métodos , Linfócitos T/citologia , Animais , Movimento Celular/fisiologia , Corantes Fluorescentes/química , Linfonodos/imunologia , Camundongos
10.
J Cell Sci ; 123(Pt 10): 1785-95, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20427320

RESUMO

Cilia and flagella are eukaryotic organelles involved in multiple cellular functions. The primary cilium is generally non motile and found in numerous vertebrate cell types where it controls key signalling pathways. Despite a common architecture, ultrastructural data suggest some differences in their organisation. Here, we report the first detailed characterisation of the ciliary pocket, a depression of the plasma membrane in which the primary cilium is rooted. This structure is found at low frequency in kidney epithelial cells (IMCD3) but is associated with virtually all primary cilia in retinal pigment epithelial cells (RPE1). Transmission and scanning electron microscopy, immunofluorescence analysis and videomicroscopy revealed that the ciliary pocket establishes closed links with the actin-based cytoskeleton and that it is enriched in active and dynamic clathrin-coated pits. The existence of the ciliary pocket was confirmed in mouse tissues bearing primary cilia (cumulus), as well as motile cilia and flagella (ependymal cells and spermatids). The ciliary pocket shares striking morphological and functional similarities with the flagellar pocket of Trypanosomatids, a trafficking-specialised membrane domain at the base of the flagellum. Our data therefore highlight the conserved role of membrane trafficking in the vicinity of cilia.


Assuntos
Actinas/metabolismo , Cílios/metabolismo , Citoesqueleto/metabolismo , Endocitose , Flagelos/metabolismo , Animais , Linhagem Celular , Movimento Celular , Cílios/patologia , Epitélio/patologia , Feminino , Fibroblastos/patologia , Flagelos/patologia , Humanos , Microdomínios da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Ovulação , Zona Pelúcida/metabolismo
11.
Blood ; 115(22): 4412-20, 2010 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-20308597

RESUMO

Cross-presentation is an essential mechanism that allows dendritic cells (DCs) to efficiently present exogenous antigens to CD8(+) T cells. Among cellular antigen sources, apoptotic cells are commonly considered as the best for cross-presentation by DCs. However, the potential of live cells as a source of antigen has been overlooked. Here we explored whether DCs were able to capture and cross-present antigens from live cells. DCs internalized cytosolic and membrane material into vesicles from metabolically labeled live cells. Using time-lapse confocal microscopy in whole spleens, we showed that DCs internalized material from live cells in vivo. After ovalbumin uptake from live cells, DCs cross-primed ovalbumin-specific naive OT-I CD8(+) T cells in vitro. Injected into mice previously transferred with naive OT-I T cells, they also cross-primed in vivo, even in the absence of endogenous DCs able to present the epitope in the recipient mice. Interestingly, DCs induced stronger natural CD8(+) T-cell responses and protection against a lethal tumor challenge after capture of antigens from live melanoma cells than from apoptotic melanoma cells. The potential for cross-presentation from live cells uncovers a new type of cellular intercommunication and must be taken into account for induction of tolerance or immunity against self, tumors, grafts, or pathogens.


Assuntos
Apresentação Cruzada , Células Dendríticas/imunologia , Animais , Apresentação do Antígeno , Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Imunidade Celular , Técnicas In Vitro , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Ovalbumina/imunologia
12.
J Cell Biol ; 183(7): 1287-98, 2008 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-19114595

RESUMO

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by alphaMbeta2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Antígeno de Macrófago 1/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Fagocitose/fisiologia , Animais , Células Cultivadas , Macrófagos/metabolismo , Camundongos , Interferência de RNA , Transfecção
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